Ribonuclease P (RNase P) catalyzes 5' end maturation of precursor tRNA (pre-tRNA) to form tRNA, an essential component of protein synthesis. RNase P is found in all domains of life, but the composition of this indispensable enzyme varies from a RNA-protein heterodimer in bacteria to a complex of three proteins in human mitochondrial RNase P (mtRNase P). These enzymes provide an ideal system for defining catalytic features that distinguish RNA- and protein-based catalysis. Furthermore, the distinct subunit compositions highlight the potential of bacterial RNase P as a novel antibiotic target. In mitochondria, mutations in (mt)tRNA and mtRNase P subunits have been linked to a number of diseases, including neurodegeneration, X-linked mental retardation, myocardial infarction, coronary artery disease as well as mitochondria dysfunction which manifests clinically as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like symptoms), progressive external opthalmoplegia and/or diabetes. Analysis of the in vivo and in vitro function of mtRNase P will provide insight into mitochondrial tRNA processing pathways and their role in mitochondria biogenesis and dysfunction. Thus, investigation of RNase P structure and function has the potential for wide-ranging impact on a variety of health issues, from improving antibacterial therapeutics to characterization of the biological pathways linked to the pathogenesis of multiple mitochondrial diseases. This proposal consists of two primary objectives. First, we propose to develop biophysical methods, including single molecule fluorescence spectroscopy and NMR spectroscopy (in collaboration with Professors Al-Hashimi and Walter) to investigate two hallmark features of large RNA molecules, such as the RNase P RNA subunit: dynamic RNA-metal interactions that exchange between diffusive, inner-sphere, and outer- sphere contacts; and conformational plasticity that is central to RNA function, including substrate recognition and catalysis. In applying these methods to bacterial RNase P we aim to: (1) explore the changes in structure and dynamics that occur in RNase P throughout the catalytic cycle; and (2) delineate the structure and interactions within proposed metal ion binding sites in RNase P. Second, we will identify the strategies employed by the newly discovered protein-based mtRNase P to achieve catalysis and substrate recognition. In particular, we explore the function of MRPP3 using mutagenesis, metal substitution and kinetic analysis to elucidate mechanistic features of this member of a novel family predicted to have metal-dependent nuclease activity. Finally, we will examine determinants of pre-tRNA recognition and the role of defects in mtRNase P processing in the pathophysiological mechanisms of human mitochondrial tRNA mutations. These studies will significantly enhance our understanding of the structure and function of these two distinct classes of RNase P enzymes and their homologues, develop methods useful for studying similar enzymes, and provide fundamental insights into the nature of biological catalysis.